Reversed-phase high performance liquid chromatography (HPLC) is generally a highly useful technique for resolution of different proteins. A problem, however, exists in the chromatography art in that on occasion, HPLC cannot resolve proteins that one would ordinarily expect to resolve on the basis of different physicochemical properties. For example, TGF.alpha.PE.sub.40 ab and PE.sub.40 ab, which differ in length by approximately 50 amino acid residues, were unexpectedly found to resist resolution by reversed-phase HPLC. Numerous attempts were made using a number of different columns under a variety of mobile phase conditions. The types of columns tried included different silica-based columns with varying length hydrocarbonaceous ligates as well as wide-pore polystyrene divinylbenzene polymeric resins. Various mobile phases at pH 2, 4.5, 7, 8.5, and 10 were evaluated, as were a number of organic modifiers. In all cases, TGF.alpha.PE.sub.40 ab could not be resolved satisfactorily from PE.sub.40 ab.
Pseudomonas aeruginosa produces the exotoxin Pseudomonas exotoxin A. The exotoxin consists of four structural domains, namely Ia, II, Ib and III. When domain Ia is cleaved off the exotoxin, the resulting protein is known as PE.sub.40. Transforming growth factor alpha (TGF.alpha.) can be genetically fused to the amino terminus of PE.sub.40 to produce the chimeric protein TGF.alpha.PE.sub.40. When cysteine residues in domain II of PE.sub.40 are deleted or substituted by non-cysteinyl amino acids, the resulting protein is PE.sub.40 ab, which when genetically fused to TGF.alpha. produces TGF.alpha.PE.sub.40 ab. The TGF.alpha. portion of the hybrid will still contain cysteine residues. Thus, PE.sub.40 ab and TGF.alpha.PE.sub.40 ab can be differentiated on the basis of presence or absence of cysteine residues.
TGF.alpha.PE.sub.40 ab is useful in treatment of certain tumor cells. Its manufacture involves a fermentation process that requires a rapid assay that can monitor the product at each step of manufacturing from fermentation to the final product. TGF.alpha.PE.sub.40 ab is an intracellular product which, in the early stages of growth, represents only a small percentage of the total amount of cellular protein. Reversed-phase HPLC was able to resolve TGF.alpha.PE.sub.40 ab from the host cell proteins, but was not able to resolve it from PE.sub.40 ab. This caused inaccurate quantitation of expression levels of TGF.alpha.PE.sub.40 ab. This additionally caused the problem of being unable to estimate recovery and yield of each step in the purification process. Although, PE.sub.40 ab and TGF.alpha.PE.sub.40 ab can be resolved by western blot analysis, this technique is impractical for monitoring commercial scale batches. Therefore, a need exists in the art for a method of assaying proteins that differ in physicochemical properties, such as size, but are not resolved by reversed-phase HPLC. The present invention meets that need by exploiting the presence or absence of cysteinyl amino acid residues between two or more proteins.